Precise pathogen detection in the presence of inhibitors

Detection of low-level pathogens is important in situations where bacterial or viral pathogens cause human illnesses, often through contaminated food or water supplies. In this context, it is important to detect pathogens in the food chain at the earliest opportunity to reduce their impact. While real-time PCR (qPCR) can be a useful tool for pathogen detection in general, it is sensitive to PCR inhibitors present in the crude samples common to food and water testing.

By contrast, digital PCR is less sensitive to inhibitors and offers a more robust testing platform. It works by partitioning each sample into thousands of independent, parallel PCR reactions. Digital PCR is not as easily affected by inhibitors than real-time PCR, because it is an endpoint reaction, and as such it is possible to more accurately and precisely measure pathogen concentration in what would be an otherwise highly inhibited sample. In addition, digital PCR provides an absolute count of pathogenic sequences, eliminating the need for reference material or a standard curve.

Measuring the absolute number of molecules in a biological sample is a relatively common need. But, this can pose some unique challenges when using standard real-time PCR. In contrast, digital PCR is not dependent on cycle thresholds or references, yielding a simple and precise method to obtain absolute quantification

Deborah Grove, PhD, Director - Genetic Analysis, Genomics Core Facility at The Huck Institutes of the Life Sciences Penn State University Park, Pennsylvania

Figure 1.Pathogen detection in the presence of an inhibitor. A water sample was mock-contaminated with a common environmental PCR inhibitor, humic acid. The data demonstrate the reduced impact on digital PCR of inhibitors that would otherwise interfere in a standard real-time PCR reaction. The gray line shows drastic real-time PCR inhibition with wider error bars, and significantly lower measured concentration, at higher levels of humic acid. In contrast, the accuracy in measurement of pathogen concentration by digital PCR (blue line) is not as affected, as demonstrated by the lower slope and tighter error bars.